Advances in gene recombinant technology have enabled a stable supply of various protein formulations. In particular, a variety of recombinant antibody drugs, which are more selective than normal drugs, have been developed and entered clinical trial in recent years.
In these recombinantly-produced physiologically active protein-containing formulations, there is a need to remove host DNA and impurities (e.g., DNA contaminants) associated with viral contamination. Under present World Health Organization (WHO) criteria, the amount of DNA in biological drugs should not exceed 100 pg DNA/dose. To meet this criteria, in general, an aqueous medium containing host cell-derived physiologically active proteins is treated by anion-exchange chromatography, hydroxyapatite chromatography or a combination thereof, for the purpose of removing DNA.
In particular, in a case where a physiologically active protein is an antibody produced, recombinantly in mammalian host cells, the aqueous medium is treated by affinity column chromatography on Protein A or G before being purified by various types of chromatography, based, on the binding property of Protein A or Protein G to IgG Fc chain.
By way of example, in JP KOHYO 5-504579, an antibody-containing aqueous medium obtained from mammalian cell culture is subjected to Protein A/G column chromatography to adsorb antibody molecules onto the column, followed by elution with an acidic solution (about 0.1 M citric acid, pH 3.0-3.5) to release the antibody molecules. The resulting acidic eluate is subjected sequentially to ion-exchange column chromatography and size exclusion column chromatography to give the purified antibody molecules.
However, these individual chromatographic processes and combinations thereof are time-, labor- and cost-consuming, as well as being complicated. Moreover, they fail to provide stable results.
Thus, the object of the present invention is to provide a simpler and less expensive method for purifying physiologically active proteins, especially antibodies, which can ensure removal of impurities such as DNA contaminants and viruses, and which can minimize a loss of physiologically active proteins.